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Abstract:

The invention provides methods for screening test compounds or toxins for
effects on cells. The invention also provides methods for determining
frequency, amplitude and kinetic profiles of cells.

Claims:

1. A method for screening a compound or environmental condition for an
effect on cells, cell aggregates, or tissue comprising: (a) applying the
cells, cell aggregates, or tissue to a colorimetric resonant reflectance
biosensor surface, a dielectric film stack biosensor surface, or a
grating-based waveguide biosensor surface; (b) contacting the cells, cell
aggregates, or tissue with the compound or environmental condition; (c)
detecting periodic or continuous peak wavelength values or effective
refractive index values during a time course; (d) analyzing the peak
wavelength values or effective refractive index values for frequency,
amplitude, or kinetic profile, or a combination thereof over the time
course; wherein a change in frequency, amplitude, or kinetic profile
after the compound or environmental condition is contacted with the
cells, cell aggregates, or tissue indicates that the compound or
environmental condition has an effect on the cells, cell aggregates, or
tissue.

2. The method of claim 1, wherein two or more concentrations of the
compound are added to one or more populations the cells, cell aggregates,
or tissue at two or more distinct locations on the biosensor surface.

4. The method of claim 3, wherein the human or mammalian induced
pluripotent stem cell line, or cells differentiated from the human or
mammalian induced pluripotent cells are cardiomyocytes.

5. The method of claim 1, wherein the peak wavelength values or effective
refractive index values are analyzed for frequency or amplitude, wherein
a decreased frequency over the time course of the assay indicates a
negative effect of the compound or environmental condition on the cells,
cell aggregates, or tissue, and wherein a decreased amplitude over the
time course of the assay indicates a negative effect of the compound or
environmental condition on the cells, cell aggregates or tissue.

6. The method of claim 2, wherein the peak wavelength values or effective
refractive index values are analyzed for frequency or amplitude, wherein
a decreased frequency with increasing compound concentration indicates a
negative effect of the compound on the cells, cell aggregates, or tissue
and wherein a decreased amplitude with increasing compound concentration
indicates a negative effect of the compound or environmental condition on
the cells, cell aggregates, or tissue.

7. The method of claim 1, wherein the peak wavelength values are analyzed
for kinetic profile, wherein a kinetic profile that moves from a positive
peak wavelength value to a negative peak wavelength value over the time
course indicates a negative effect of the compound or environmental
condition on the cells, cell aggregates, or tissue.

8. The method of claim 2, wherein the peak wavelength values are analyzed
for kinetic profile, wherein a kinetic profile that moves from a positive
peak wavelength value to a negative peak wavelength value with increasing
concentration of the compound indicates a negative effect of the compound
or environmental condition on the cells, cell aggregates, or tissue.

9. The method of claim 1, wherein the compound is a drug, a calcium
channel blocker, a β-adrenoreceptor agonist, an
α-adrenoreceptor agonist, test reagent, a polypeptide, a
polynucleotide, a modifier of a hERG channel, or a toxin.

10. The method of claim 1, wherein the cell aggregates are embroid
bodies.

11. A method for reducing the risk of pharmacological agent toxicity in a
subject, comprising: (a) contacting one or more cells differentiated from
an induced pluripotent stem cell line generated from the subject with a
dose of a pharmacological agent; (b) assaying the contacted one or more
cells for toxicity comprising: (i) applying the cells to a colorimetric
resonant reflectance biosensor surface, a dielectric film stack biosensor
surface, or a grating-based waveguide biosensor surface; (ii) contacting
the cells with the pharmacological agent; (iii) detecting periodic or
continuous peak wavelength values or effective refractive index values
during a time course; (iv) analyzing the peak wavelength values or
effective refractive index values for frequency, amplitude, or kinetic
profile or a combination thereof over the time course; wherein a negative
change in frequency, amplitude, or kinetic profile after the
pharmacological agent is contacted with the cells indicates that the
pharmacological agent has a negative toxicity effect on the cells; (c)
prescribing or administering the pharmacological agent to the subject
only if the pharmacological agent does not have a negative toxicity
effect on the contacted cells, thereby reducing the risk of
pharmacological toxicity in a subject.

12. A method for reducing the risk of pharmacological agent toxicity in a
subject, comprising: (a) contacting one or more cell populations
differentiated from an induced pluripotent stem cell line generated from
the subject with two or more dose concentrations of a pharmacological
agent; (b) assaying the contacted one or more cell populations for
toxicity comprising: (i) applying the one or more cell populations to a
colorimetric resonant reflectance biosensor surface, a dielectric film
stack biosensor surface or a grating-based waveguide biosensor surface;
(ii) contacting the one or more cell populations with two of more
concentrations the pharmacological agent; (iii) detecting one or more
peak wavelength values or effective refractive index values for each
concentration of the pharmacological agent; (iv) analyzing the peak
wavelength values or effective refractive index values for frequency,
amplitude, or kinetic profile or a combination thereof for each
concentration of the pharmacological agent; wherein a negative change in
frequency, amplitude, or kinetic profile after the pharmacological agent
is contacted with the cells indicates that the pharmacological agent
concentration has a negative toxicity effect on the cells; (c)
prescribing or administering the pharmacological agent to the subject
only if the pharmacological agent concentration does not have a negative
toxicity effect in the contacted cells, thereby reducing the risk of
pharmacological toxicity in a subject.

13. A method of screening a compound for neutralizing activity on a toxin
or negative environmental condition comprising: (a) applying cells, cell
aggregates, or tissue to a colorimetric resonant reflectance biosensor
surface, a dielectric film stack biosensor surface, or a grating-based
waveguide biosensor surface; (b) contacting the cells, cell aggregates,
or tissue with the toxin or negative environmental condition and the
compound; (c) detecting periodic or continuous peak wavelength values or
effective refractive index values during a time course; (d) analyzing
peak wavelength values or effective refractive index values for
frequency, amplitude, or kinetic profile or a combination thereof over
the time course; wherein a positive change in frequency, amplitude, or
kinetic profile after the compound is contacted with the cells, cell
aggregates, or tissue indicates that the compound has a neutralizing
effect on the toxin or negative environmental condition.

14. A method of screening a compound for neutralizing activity on a toxin
or negative environmental condition comprising: (a) applying one or more
cells, cell aggregates, or tissue populations to a colorimetric resonant
reflectance biosensor surface, a dielectric film stack biosensor surface,
or a grating-based waveguide biosensor surface; (b) contacting the one or
more cells, cell aggregates, or tissue populations with the toxin or
negative environmental condition and the compound at two or more compound
concentrations; (c) detecting periodic or continuous peak wavelength
values or effective refractive index values during a time course for each
compound concentration; (d) analyzing peak wavelength values or effective
refractive index values for frequency, amplitude, or kinetic profile or a
combination thereof for each compound concentration over the time course;
wherein a positive change in frequency, amplitude, or kinetic profile
after the compound is contacted with the cells, cell aggregates, or
tissue indicates that the compound has a neutralizing effect on the toxin
or negative environmental condition.

15. The method of claim 1, wherein said contacting step further includes
contacting the cells, cell aggregates, or tissue with at least one second
compound or at least one second environmental condition in the presence
of the first compound or the first environmental condition.

16. A method of screening a test toxin for a signature kinetic profile to
determine a class or subclass of the test toxin comprising: (a) applying
cells, cell aggregates, or tissue to a colorimetric resonant reflectance
biosensor surface, a dielectric film stack biosensor surface, or a
grating-based waveguide biosensor surface; (b) contacting the cells, cell
aggregates, or tissue with the test toxin; (c) detecting periodic or
continuous peak wavelength values or effective refractive index values
during a time course of the assay; (d) analyzing the peak wavelength
values or effective refractive index values for frequency, amplitude, or
kinetic profile or a combination thereof over the time course of the
assay to generate a signature kinetic profile of the test toxin's effects
on the cells, cell aggregates, or tissue; and (e) comparing the signature
kinetic profile of the test toxin to signature kinetic profiles of known
toxins, wherein the test toxin is placed into a class or subclass of
toxins having a similar signature kinetic profile as the test toxin.

17. A method for determining the effect of a test compound or
environmental condition on the sinus rhythm of cardiomyocytes
compromising: (a) applying the cardiomyocytes to a colorimetric resonant
reflectance biosensor surface, a dielectric film stack biosensor surface,
or a grating-based waveguide biosensor surface; (b) contacting the
cardiomyocytes with the compound or environmental condition; (c)
detecting periodic or continuous peak wavelength values or effective
refractive index values during a time course; (d) analyzing the peak
wavelength values or effective refractive index values for sinus rhythm
over the time course; wherein a change in the sinus rhythm after the
compound or environmental condition is contacted with the cardiomyocytes
indicates that the compound or environmental condition has an effect on
the sinus rhythm of the cardiomyocytes.

18. The method of claim 17, wherein the effect of the test compound or
environmental condition on the sinus rhythm is a prolongation or
shortening of the QT interval.

19. A method for determining a beat or burst pattern of cardiac or
neuronal cells, cardiac or neuron cell aggregates, or cardiac or neuronal
tissue comprising: (a) applying the cells, cell aggregates, or tissue to
a colorimetric resonant reflectance biosensor surface, a dielectric film
stack biosensor surface, or a grating-based waveguide biosensor surface;
(b) detecting periodic or continuous peak wavelength values or effective
refractive index values during a time course; (c) analyzing the peak
wavelength values or effective refractive index values for frequency,
amplitude, or kinetic profile, or a combination thereof over the time
course; wherein a beat or burst pattern of the cardiac or neuronal cells,
cardiac or neuron cell aggregates, or cardiac or neuronal tissue is
determined.

20. The method of claim 19, wherein one or more compounds are added to
the cells, cell aggregates, or tissue before or after they are applied to
the biosensor surface.

Description:

PRIORITY

[0001] This application claims the benefit of U.S. Ser. No. 61/317,995,
which was filed on Mar. 26, 2010, U.S. Ser. No. 61/323,076, which was
filed on Apr. 12, 2010, and U.S. Ser. No. 61/363,824, which was filed on
Jul. 13, 2010. All of these applications are incorporated by reference
herein in their entirety.

SUMMARY OF THE INVENTION

[0002] One embodiment of the invention provides a method for screening a
compound or environmental condition for an effect on cells, cell
aggregates, or tissue. The method comprises applying the cells, cell
aggregates, or tissue to a colorimetric resonant reflectance biosensor
surface, a dielectric film stack biosensor surface, or a grating-based
waveguide biosensor surface. The cells, cell aggregates, or tissue are
contacted with the compound or environmental condition and periodic or
continuous peak wavelength values or effective refractive index values
are detected during a time course. Peak wavelength values or effective
refractive index values are analyzed for frequency, amplitude, or kinetic
profile, or a combination thereof over the time course. A change in
frequency, amplitude, or kinetic profile after the compound or
environmental condition is contacted with the cells, cell aggregates, or
tissue indicates that the compound or environmental condition has an
effect on the cells, cell aggregates, or tissue. Optionally, two or more
concentrations of the compound can be added to one or more populations
the cells, cell aggregates, or tissue at two or more distinct locations
on the biosensor surface. The cells can be stem cells, human or mammalian
induced pluripotent stem cells, cells differentiated from the human or
mammalian induced pluripotent cells, neural stem cells, neurons,
cardiomyocyte stem cells, cardiomyocytes, hepatic stem cells, hepatocytes
or combinations thereof. The human or mammalian induced pluripotent stem
cell line or cells differentiated from the human or mammalian induced
pluripotent cells can be cardiomyocytes. The peak wavelength values or
effective refractive index values can be analyzed for frequency or
amplitude, wherein a decreased frequency over the time course of the
assay indicates a negative effect of the compound or environmental
condition on the cells, cell aggregates, or tissue, and wherein a
decreased amplitude over the time course of the assay indicates a
negative effect of the compound or environmental condition on the cells,
cell aggregates or tissue. The peak wavelength values or effective
refractive index values can be analyzed for frequency or amplitude,
wherein a decreased frequency with increasing compound concentration
indicates a negative effect of the compound on the cells, cell
aggregates, or tissue and wherein a decreased amplitude with increasing
compound concentration indicates a negative effect of the compound or
environmental condition on the cells, cell aggregates, or tissue. The
peak wavelength values can be analyzed for kinetic profile, wherein a
kinetic profile that moves from a positive peak wavelength value to a
negative peak wavelength value over the time course indicates a negative
effect of the compound or environmental condition on the cells, cell
aggregates, or tissue. The peak wavelength values can be analyzed for
kinetic profile, wherein a kinetic profile that moves from a positive
peak wavelength value to a negative peak wavelength value with increasing
concentration of the compound indicates a negative effect of the compound
or environmental condition on the cells, cell aggregates, or tissue. The
compound can be a drug, a calcium channel blocker, a
β-adrenoreceptor agonist, an α-adrenoreceptor agonist, a test
reagent, a polypeptide, a polynucleotide, a modifier of a hERG channel,
or a toxin. The cell aggregates can be embroid bodies. The cells, cell
aggregates, or tissue can be further contacted with at least one second
compound or second environmental condition in the presence of the first
compound or first environmental condition.

[0003] Another embodiment of the invention provides a method for reducing
the risk of pharmacological agent toxicity in a subject. The method
comprises contacting one or more cells differentiated from an induced
pluripotent stem cell line generated from the subject with a dose of a
pharmacological agent. The contacted one or more cells are assayed for
toxicity by applying the cells to a colorimetric resonant reflectance
biosensor surface, a dielectric film stack biosensor surface, or a
grating-based waveguide biosensor surface and contacting the cells with
the pharmacological agent. Periodic or continuous peak wavelength values
or effective refractive index values are detected during a time course.
The peak wavelength values or effective refractive index values are
analyzed for frequency, amplitude, or kinetic profile or a combination
thereof over the time course. A negative change in frequency, amplitude,
or kinetic profile after the pharmacological agent is contacted with the
cells indicates that the pharmacological agent has a negative toxicity
effect on the cells. The pharmacological agent is prescribed or
administered to the subject only if the pharmacological agent does not
have a negative toxicity effect on the contacted cells, thereby reducing
the risk of pharmacological toxicity in a subject.

[0004] Even another embodiment of the invention provides a method for
reducing the risk of pharmacological agent toxicity in a subject. The
method comprises contacting one or more cell populations differentiated
from an induced pluripotent stem cell line generated from the subject
with two or more dose concentrations of a pharmacological agent. The
contacted one or more cell populations are assayed for toxicity by
applying the one or more cell populations to a colorimetric resonant
reflectance biosensor surface, a dielectric film stack biosensor surface,
or a grating-based waveguide biosensor surface and contacting the one or
more cell populations with two of more concentrations the pharmacological
agent. One or more peak wavelength values or effective refractive index
values are detected for each concentration of the pharmacological agent.
The peak wavelength values or effective refractive index values are
analyzed for frequency, amplitude, or kinetic profile or a combination
thereof for each concentration of the pharmacological agent. A negative
change in frequency, amplitude, or kinetic profile after the
pharmacological agent is contacted with the cells indicates that the
pharmacological agent concentration has a negative toxicity effect on the
cells. The pharmacological agent is prescribed or administered to the
subject only if the pharmacological agent concentration does not have a
negative toxicity effect in the contacted cells, thereby reducing the
risk of pharmacological toxicity in a subject.

[0005] Still another embodiment of the invention provides a method of
screening a compound for neutralizing activity on a toxin or negative
environmental condition. The method comprises applying cells, cell
aggregates, or tissue to a colorimetric resonant reflectance biosensor
surface, a dielectric film stack biosensor surface, or a grating-based
waveguide biosensor surface and contacting the cells, cell aggregates, or
tissue with the toxin or negative environmental condition and the
compound. Periodic or continuous peak wavelength values or effective
refractive index values are detected during a time course. The peak
wavelength values or effective refractive index values are analyzed for
frequency, amplitude, or kinetic profile or a combination thereof over
the time course. A positive change in frequency, amplitude, or kinetic
profile after the compound is contacted with the cells, cell aggregates,
or tissue indicates that the compound has a neutralizing effect on the
toxin or negative environmental condition.

[0006] Yet another embodiment of the invention provides a method of
screening a compound for neutralizing activity on a toxin or negative
environmental condition. One or more cells, cell aggregates, or tissue
populations are applied to a colorimetric resonant reflectance biosensor
surface, a dielectric film stack biosensor surface, or a grating-based
waveguide biosensor surface and contacted with the toxin or negative
environmental condition and the compound at two or more compound
concentrations. Periodic or continuous peak wavelength values or
effective refractive index values are detected during a time course for
each compound concentration. Peak wavelength values or effective
refractive index values are analyzed for frequency, amplitude, or kinetic
profile or a combination thereof for each compound concentration over the
time course. A positive change in frequency, amplitude, or kinetic
profile after the compound is contacted with the cells, cell aggregates,
or tissue indicates that the compound has a neutralizing effect on the
toxin.

[0007] Another embodiment of the invention provides a method of screening
a test toxin for a signature kinetic profile to determine a class or
subclass of the test toxin. The method comprises applying cells, cell
aggregates, or tissue to a colorimetric resonant reflectance biosensor
surface, a dielectric film stack biosensor surface, or a grating-based
waveguide biosensor surface and contacting the cells, cell aggregates, or
tissue with the test toxin. Periodic or continuous peak wavelength values
or effective refractive index values are detected during a time course of
the assay. The peak wavelength values or effective refractive index
values are analyzed for frequency, amplitude, or kinetic profile or a
combination thereof over the time course of the assay to generate a
signature kinetic profile of the test toxin's effects on the cells, cell
aggregates, or tissue. The signature kinetic profile of the test toxin is
compared to signature kinetic profiles of known toxins, wherein the test
toxin is placed into a class or subclass of toxins having a similar
signature kinetic profile as the test toxin.

[0008] Even another embodiment of the invention provides a method for
determining the effect of a test compound or environmental condition on
the sinus rhythm of cardiomyocytes. The method comprises applying the
cardiomyocytes to a colorimetric resonant reflectance biosensor surface,
a dielectric film stack biosensor surface, or a grating-based waveguide
biosensor surface and contacting the cardiomyocytes with the compound or
environmental condition. Periodic or continuous peak wavelength values or
effective refractive index values are detected during a time course. The
peak wavelength values or effective refractive index values are analyzed
for sinus rhythm over the time course. A change in the sinus rhythm after
the compound or environmental condition is contacted with the
cardiomyocytes indicates that the compound or environmental condition has
an effect on the sinus rhythm of the cardiomyocytes. The effect of the
test compound or environmental condition on the sinus rhythm can be a
prolongation or shortening of the QT interval.

[0009] Another embodiment of the invention provides a method for
determining a beat or burst pattern of cardiac or neuronal cells, cardiac
or neuron cell aggregates, or cardiac or neuronal tissue. The method
comprises applying the cells, cell aggregates, or tissue to a
colorimetric resonant reflectance biosensor surface, a dielectric film
stack biosensor surface, or a grating-based waveguide biosensor surface
and detecting periodic or continuous peak wavelength values or effective
refractive index values during a time course. The peak wavelength values
or effective refractive index values are analyzed for frequency,
amplitude, or kinetic profile, or a combination thereof over the time
course. A beat or burst pattern of the cardiac or neuronal cells, cardiac
or neuron cell aggregates, or cardiac or neuronal tissue is determined.
Optionally, one or more compounds are added to the cells, cell
aggregates, or tissue before or after they are applied to the biosensor
surface.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]FIG. 1 demonstrates the kinetic profile of two toxins on Vero
cells.

[0029] FIG. 17A-D shows the effect of amitriptyline on cardiomyocytes.
FIG. 17A shows the cardiomyocytes prior to the addition of the
amitriptyline. FIGS. 17B, 17C, and 17D show the cardiomyocytes over time
at 6 minutes, 10 minutes, and 15 minutes, respectively, after the
addition of amitriptyline.

DETAILED DESCRIPTION OF THE INVENTION

[0030] As used herein, the singular forms "a," "an," and "the" include
plural referents unless the context clearly dictates otherwise.

Biosensors

[0031] Biosensors of the invention can be colorimetric resonant
reflectance biosensors or grating-based waveguide biosensors. See e.g.,
Cunningham et al., "Colorimetric resonant reflection as a direct
biochemical assay technique," Sensors and Actuators B, Volume 81, p.
316-328, Jan. 5, 2002; U.S. Pat. Publ. No. 2004/0091397; U.S. Pat. No.
7,094,595; U.S. Pat. No. 7,264,973. Colorimetric resonant biosensors are
not surface plasmon resonant (SPR) biosensors. SPR biosensors have a thin
metal layer, such as silver, gold, copper, aluminum, sodium, and indium.
The metal must have conduction band electrons capable of resonating with
light at a suitable wavelength. A SPR biosensor surface exposed to light
must be pure metal. Oxides, sulfides and other films interfere with SPR.
Colorimetric resonant biosensors do not have a metal layer, rather they
have a dielectric coating of high refractive index material, such as zinc
sulfide, titanium dioxide, tantalum oxide, and silicon nitride.

[0032] Biosensors of the invention can also be dielectric film stack
biosensors (see e.g., U.S. Pat. No. 6,320,991), diffraction grating
biosensors (see e.g., U.S. Pat. Nos. 5,955,378; 6,100,991) and
diffraction anomaly biosensors (see e.g., U.S. Pat. No. 5,925,878;
RE37,473). Dielectric film stack biosensors comprise a stack of
dielectric layers formed on a substrate having a grooved surface or
grating surface (see e.g., U.S. Pat. No. 6,320,991). The biosensor
receives light and, for at least one angle of incidence, a portion of the
light propagates within the dielectric layers. The parameters of a sample
medium are determined by detecting shifts in optical anomalies, i.e.,
shifts in a resonance peak or notch. Shifts in optical anomalies can be
detected as either a shift in a resonance angle or a shift in resonance
wavelength.

[0033] Other biosensors that can be used with the methods of the invention
include grating-based waveguide biosensors, which are described in, e.g.,
U.S. Pat. No. 5,738,825. A grating-based waveguide biosensor comprises a
waveguiding film and a diffraction grating that incouples an incident
light field into the waveguiding film to generate a diffracted light
field. A change in the effective refractive index of the waveguiding film
is detected. Devices where the wave must be transported a significant
distance within the device, such as grating-based waveguide biosensors,
lack the spatial resolution of colorimetric resonant reflection
biosensors.

[0034] A colorimetric resonant reflectance biosensor allows biochemical
interactions to be measured on the biosensor's surface without the use of
fluorescent tags, colorimetric labels or any other type of detection tag
or detection label. A biosensor surface contains an optical structure
that, when illuminated with collimated and/or white light, is designed to
reflect or transmit only a narrow band of wavelengths ("a resonant
grating effect"). For reflection the narrow wavelength band is described
as a wavelength "peak." For transmission the narrow wavelength band is
described as a wavelength "dip." The "peak wavelength value" (PWV)
changes when materials, such as biological materials, are deposited or
removed from the biosensor surface. Wavelength dips can also be detected.
A readout instrument is used to illuminate distinct locations on a
biosensor surface with collimated and/or white light, and to collect
reflected light. The collected light is gathered into a wavelength
spectrometer for determination of a PWV.

[0035] Wherever the changes in PWV is discussed herein, it is understood
that shifts in resonance angle, shifts in resonance wavelength, and
changes in effective refractive index can be substituted depending upon
the type of biosensor used. Additionally, where colorimetric resonant
reflectance biosensors are discussed herein, it is understood that
dielectric film stack biosensors, diffraction grating biosensors,
diffraction anomaly biosensors, and grating-based waveguide biosensors
can be substituted.

[0036] A detection system can comprise a biosensor, a light source that
directs light to the biosensor, and a detector that detects light
reflected from the biosensor. In one embodiment, it is possible to
simplify the readout instrumentation by the application of a filter so
that only positive results over a determined threshold trigger a
detection.

[0037] A light source can illuminate a colorimetric resonant reflectance
biosensor from its top surface, i.e., the surface to which cells are
applied or from its bottom surface. By measuring the shift in resonant
wavelength at each distinct location of a biosensor, it is possible to
determine which distinct locations have mass bound to or associated with
them. The extent of the shift can be used to determine, e.g., the amount
of ligands in a test sample or the chemical affinity between one or more
specific binding substances and the ligands of the test sample. The
extent of shift can also be used to detect small changes in mass on the
sensor surface.

[0038] A biosensor can be illuminated twice. The first measurement
determines the reflectance spectra of one or more distinct locations of a
biosensor array with cells immobilized on the biosensor. The second
measurement determines the reflectance spectra after one or more
compounds are applied to a biosensor or a change in environmental
conditions is made. The difference in peak wavelength between these two
measurements is a measurement of the effect of the compounds or
environmental conditions on the cells. This method of illumination can
control for small nonuniformities in a surface of a biosensor that can
result in regions with slight variations in the peak resonant wavelength.
This method can also control for varying concentrations or molecular
weights of cells on a biosensor.

[0039] A biosensor can be illuminated two or more times at two or more
time points to create periodic peak wavelength readings (or other
readings, e.g., shift in resonant angle readings, shift in wavelength
readings, or refractive index readings). Alternatively, a biosensor can
be continuously illuminated and readings collected continuously. A time
course of an assay can be about 1/100, 1/10 or 1/2 of second, 1, 2, 5,
10, 20, 30, 45 or 60 seconds, 1, 2, 3, 4, 5, 10, 20, or 60 minutes, 2, 3,
4, 5, 12, 24, 36, 48, 72 hours or more.

[0040] Cells such as primary cells or stem cells can be immobilized to the
biosensor by one or more ligands. In one embodiment of the invention,
cells are immobilized to the biosensor through a reaction with
extracellular matrix ligands. Integrins are cell surface receptors that
interact with the extracellular matrix (ECM) and mediate intracellular
signals. Integrins are responsible for cytoskeletal organization,
cellular motility, regulation of the cell cycle, regulation of cellular
of integrin affinity, attachment of cells to viruses, attachment of cells
to other cells or ECM. Integrins are also responsible for signal
transduction, a process whereby the cell transforms one kind of signal or
stimulus into another intracellularly and intercellularly. Integrins can
transduce information from the ECM to the cell and information from the
cell to other cells (e.g., via integrins on the other cells) or to the
ECM. A list of integrins and their ECM ligands can be found in Takada et
al. Genome Biology 8:215 (2007) as shown in Table 1.

[0042] Yet other cell surface receptors can include, but are not limited
to those that can interact with the ECM include cluster of
differentiation (CD) molecules. CD molecules act in a variety of ways and
often act as receptors or ligands for the cell. Other cell surface
receptors that interact with ECM include cadherins, adherins, and
selectins.

[0043] The ECM has many functions including providing support and
anchorage for cells, segregation of tissue from one another, and
regulation of intracellular communications. The ECM is composed of
fibrous proteins and glycosaminoglycans. Glycosaminoglycans are
carbohydrate polymers that are usually attached to the ECM proteins to
form proteoglycans (including, e.g., heparin sulfate proteoglycans,
chondroitin sulfate proteoglycans, karatin sulfate proteoglycans). A
glycosaminoglycan that is not found as a proteoglycan is hyaluronic acid.
ECM proteins include, for example, collagen (including fibrillar, facit,
short chain, basement membrane and other forms of collagen), fibronectin,
elastin, and laminin (see Table 1 for additional examples of ECM
proteins). ECM ligands useful herein include ECM proteins and/or peptide
fragments thereof (e.g. RGD-containing peptide fragments of fibronectin
or peptide fragments of collagen), glycosaminoglycans, proteoglycans, and
hyaluronic acid.

[0044] "Specifically binds," "specifically bind" or "specific for" means
that a cell surface receptor, e.g., an integrin or focal adhesion
protein, etc., binds to a cognate extracellular matrix ligand, with
greater affinity than to other, non-specific molecules. A non-specific
molecule does not substantially bind to the cell receptor. For example,
the integrin α4/β1 specifically binds the ECM ligand
fibronectin, but does not specifically bind the non-specific ECM ligands
collagen or laminin. In one embodiment of the invention, specific binding
of a cell surface receptor to an extracellular matrix ligand, wherein the
extracellular matrix ligand is immobilized to a surface, e.g., a
biosensor surface, will immobilize the cell to the extracellular matrix
ligand and therefore to the surface such that the cell is not washed from
the surface by normal cell assay washing procedures.

[0045] By specifically immobilizing cells to a biosensor surface through
binding of cell surface receptors, such as integrins, to ECM ligands that
are immobilized to the biosensor, the binding of the cells to the
biosensor and the cells' response to stimuli can be dramatically altered
as compared to cells that are non-specifically immobilized to a biosensor
surface. Although not required, detection of response of cells to stimuli
can be greatly enhanced or augmented where cells are immobilized to a
biosensor via ECM ligand binding. In one embodiment of the invention, the
cells are in a serum-free medium. A serum-free medium contains about 10,
5, 4, 3, 2, 1, 0.5% or less serum. A serum-free medium can comprise about
0% serum or about 0% to about 1% serum. In one embodiment of the
invention cells are added to a biosensor surface where one or more types
of ECM ligands have been immobilized to the biosensor surface. In another
embodiment of the invention, cells are combined with one or more types of
ECM ligands and then added to the surface of a biosensor.

[0046] In one embodiment of the invention, an ECM ligand is purified. A
purified ECM ligand is an ECM ligand preparation that is substantially
free of cellular material, other types of ECM ligands, chemical
precursors, chemicals used in preparation of the ECM ligand, or
combinations thereof. An ECM ligand preparation that is substantially
free of other types of ECM ligands, cellular material, culture medium,
chemical precursors, chemicals used in preparation of the ECM ligand,
etc., has less than about 30%, 20%, 10%, 5%, 1% or more of other ECM
ligands, culture medium, chemical precursors, and/or other chemicals used
in preparation. Therefore, a purified ECM ligand is about 70%, 80%, 90%,
95%, 99% or more pure. A purified ECM ligand does not include unpurified
or semi-purified preparations or mixtures of ECM ligands that are less
than 70% pure, e.g., fetal bovine serum. In one embodiment of the
invention, ECM ligands are not purified and comprise a mixture of ECM
proteins and non-ECM proteins. Examples of non-purified ECM ligand
preparations include fetal bovine serum, bovine serum albumin, and
ovalbumin.

[0047] A biosensor of the invention can comprise an inner surface, for
example, a bottom surface of a liquid-containing vessel. A
liquid-containing vessel can be, for example, a microtiter plate well, a
test tube, a petri dish, or a microfluidic channel. One embodiment of
this invention is a biosensor that is incorporated into any type of
microtiter plate. For example, a biosensor can be incorporated into the
bottom surface of a microtiter plate by assembling the walls of the
reaction vessels over the biosensor surface, so that each reaction "spot"
can be exposed to a distinct test sample. Therefore, each individual
microtiter plate well can act as a separate reaction vessel. Separate
chemical reactions can, therefore, occur within adjacent wells without
intermixing reaction fluids and chemically distinct test solutions can be
applied to individual wells.

Cell Assays

[0048] Assays of the invention can provide more information than just a
readout on cell death versus cells remaining alive. Assays of the
invention can provide frequency, rate and kinetic profile information for
cells in culture. In particular, assays of the invention can be used to
measure the frequency, rate, and kinetic profile of beating
cardiomyocytes or bursting neurons in culture. The ability of assays of
the invention to measure the frequency and rate of beating cardiomyocytes
or bursting neurons represents a new method for measuring cytotoxic or
other effects. The `beating` phenomenon in cardiomyocytes is typically
measured one cell at a time using patch clamp methodology that measures
the opening and closing of the hERG channel--a potassium ion channel that
mediates the beating phenomenon. When the hERG channel is compromised,
such as by an inhibitor of the channel, a long QT syndrome can develop,
often leading to death. The assays of the invention allow for beating to
be measured, not just simply the voltage potential changes across one or
a few channels in a patch clamp system. Thus, the assays of the invention
allow for detection of changes in beating frequency and beating rate via
the hERG channel. Additionally, at the same time alternative ion channels
to be measured, as an orthogonal approach to patch clamp. Assays of the
invention also allow for less specific cytotoxicity to be measured on
beating or bursting cells through the ability to monitor changes in cell
adhesion and morphology in addition to the beating or bursting phenotype.

[0049] Burst or spike periods of neuronal cells can be detected with the
methods of the invention. Input-driven or intrinsic bursting of neurons
can be determined. Specific patterns that can be detected include, for
example, tonic or regular spiking by neurons that are constantly active
(e.g., interneurons), phasic bursting by neurons that fire in bursts, and
fast spiking by neurons with high firing rates (e.g., cortical inhibitory
interneurons, cells of the globus pallidus, retinal ganglion cells).
Therefore, the rate, frequency, and kinetic profile of neuronal cells
bursting or spiking in culture can be determined with the assays of the
invention. Furthermore, the effect of compounds on these burst or spike
patterns can be detected with methods of the invention.

[0050] The rate, frequency and kinetic profile can be detected in real
time using a high speed, high resolution instrument, such as the
BIND® READER (i.e., a colorimetric resonant reflectance biosensor
system), and corresponding algorithms to quantify data. See, e.g., U.S.
Pat. Nos. 7,422,891; 7,327,454, 7,301,628, 7,292,336; 7,170,599;
7,158,230; 7,142,296; 7,118,710. Additionally, cells and their
differential morphological and adhesional responses to stimuli can be
detected in real time with these methods.

[0051] The invention provides methods for screening a compound for an
effect on cells, cell aggregates or tissues. For example, the rate,
frequency, kinetic profile, or a combination thereof can be determined
for any kind of cells exposed to any type of compound, compounds,
environmental condition, environmental conditions, or combinations
thereof. Cells, cell aggregates, or tissue are applied to the surface of
a colorimetric resonant reflectance biosensor surface and one or more
test compounds or environmental conditions are added to the cells, cell
aggregates, or tissue. The compounds or environmental conditions can be
added to the cells, cell aggregates or tissue prior to the cells, cell
aggregates or tissue being applied to the biosensor surface. The PWV (or
effective refractive index) of the cells, cell aggregates, or tissue is
monitored over time. The PWV can be monitored before the compound or
environmental conditions is added, while the compound or environmental
condition is being added, after the compound or environmental condition
is added and any combination thereof. A PWV reading (or other reading)
can be taken about 1, 2, 3, 4, 5, 10, 20 or more times a second (or any
range between about 1 and 20 times a second). A PWV reading can be taken
about every 2, 5, 10, 20, 30, 45 or 60 seconds (or any range between
about 2 and 60 seconds). A PWV reading can be taken about every 1, 2, 3,
4, 5, 10, 20, or 60 minutes (or any range between about 1 and 60
minutes).

Frequency

[0052] Where the cells are cardiomyocytes, the cells will beat in culture
and the beating cells can generate a PWV pattern (alternating
positive-negative PWV shift) or effective refractive index pattern that
reveals the beating rate or frequency of the cells. See FIG. 12. The
length of time between each beat can be determined. The effect of a
compound, extracellular matrix, or environmental condition (e.g., salt
concentration, buffer type, media type, serum type, temperature, oxygen
concentration) on the frequency of the beating of the cells can be
determined.

[0053] Where the cells are neurons, the cells will burst/spike in culture
and the and the bursting/spiking cells can generate a PWV pattern or
effective refractive index pattern that reveals the bursting/spiking rate
or frequency of the bursting/spiking. The length of time between each
burst or spike can be determined. The effect of a compound, extracellular
matrix, or environmental condition on the frequency of the bursting or
spiking of the cells can be determined.

Amplitude

[0054] Where the cells are cardiomyocytes, the cells can generate a PWV
pattern or effective refractive index pattern that reveals the strength
of the cardiomyocyte beating. This is an amplitude reading. In FIG.
10A-B, cardiomyocytes are treated with either buffer or doxorubicin.
Where the cardiomyocytes are treated with buffer, the amplitude of the
beating becomes stronger over time. That is, there is Y axis spread of
the PWV reading grows larger over time as the cells beat stronger in
culture. In some cases, for example, a toxin might cause a decrease the
amplitude of the PWV reading over time, while a buffer or beneficial
compound might retain or increase the amplitude of the PWV reading over
time.

[0055] Where the cells are neurons, the cells can generate a PWV pattern
or effective refractive index pattern that reveals the strength of the
bursts or spikes. This is an amplitude reading.

Kinetic Profile

[0056] The kinetic profile is a collection of about 2, 5, 10, 20, 50, 100,
250, 500, 1,000 or more PWVs (or effective index values) of a cell
population taken over time (about 1, 5, 10, 30, 60 seconds, about 1, 2,
3, 4, 5, 10, 20, 40, 60 or more minutes). The kinetic profile reveals
changes in PWV over time and represents a unique signature of the test
compound or environmental condition. For example, where the test compound
is a toxin, the PWVs may decline over time as the cells become weaker and
then die. Where the compound is neutral or provides a benefit to the
cells the PWV over time may increase, indicating a strengthening or
growing of the cells. A kinetic profile can also be PWVs of a cell
population taken for two or more differing concentrations of a test
compound. The kinetic profile reveals changes in PWV over differing
concentrations and represents a unique signature of the test compound or
environmental condition.

Cells

[0057] The assays of the invention can be used with any cells including,
for example human or mammalian embryonic or human or mammalian adult stem
cells and induced pluripotent stem cells. Induced pluripotent stem cells
are pluripotent stem cells that are artificially produced from
non-pluripotent cells, such as adult somatic cells, by inducing forced
expression of certain genes. The induced pluripotent stem cells can be,
for example, neurons, neural stem cells, cardiomyocytes, teratomas, or
embryoid bodies. Other cells that can be used include, for example,
cardiomyocytes, hepatocytes, neurons or combinations thereof including,
for example, combinations or mixtures of hepatocytes and cardiomyocytes.
A neuron can be any type of neuron, including, for example, type I
neurons, type II neurons, interneurons, basket cells, Betz cells, medium
spiny neurons, Purkinje cells, pyramidal cells, Renshaw cells, granule
cells, anterior horn cells, or motorneurons.

Methods of Screening Cells

[0058] One embodiment of the invention provides methods for screening a
compound or environmental condition for an effect on cells, cell
aggregates, or tissue. Cells, cell aggregates, or tissue are applied to a
colorimetric resonant reflectance biosensor (or other biosensor) surface.
The cells, cell aggregates, or tissue are contacted with a test compound
or environmental condition. Periodic or continuous peak wavelength values
are determined and recorded during a time course of the assay. The peak
wavelength values are analyzed for frequency, amplitude, or kinetic
profile or a combination thereof over the time course of the assay. A
change in frequency, amplitude, or kinetic profile after the compound or
environmental condition is contacted with the cells, cell aggregates, or
tissue indicates that the compound or environmental condition has an
effect on the cells, cell aggregates, or tissue. Two or more
concentrations of the compound can be added to one or more populations
the cells, cell aggregates, or tissue at one or more distinct locations
on the biosensor surface. Where the one or more cell, cell aggregate or
tissue populations comprise two or more populations (e.g., 2, 3, 4, 5,
10, 15, 20, 100, 250, or more) the populations may be the same or
different.

[0059] A decreased frequency over the time course of the assay can
indicate a negative effect of the compound or environmental condition on
the cells and a decreased amplitude over the time course of the assay can
indicate a negative effect of the compound or environmental condition on
the cells. A negative effect can be a weakening of the cells or death of
the cells. A decreased frequency with increasing compound concentration
can indicate a negative effect of the compound on the cells and a
decreased amplitude with increasing compound concentration can indicate a
negative effect of the compound or environmental condition on the cells.

[0060] An increased frequency over the time course of the assay can
indicate a neutral or positive effect of the compound or environmental
condition on the cells and an increase in amplitude over the time course
of the assay can indicate a neutral or positive effect of the compound or
environmental condition on the cells. A positive effect or a neutral
effect can be cells strengthening, growing, or multiplying. An increase
in frequency with increasing compound concentration can indicate a
neutral or positive effect of the compound on the cells and an increase
in amplitude with increasing compound concentration can indicate a
neutral or positive effect of the compound or environmental condition on
the cells.

[0061] The peak wavelength values can be analyzed for kinetic profile,
wherein a kinetic profile that moves from a positive peak wavelength
value to a negative peak wavelength value over the time course of the
assay can indicate a negative effect of the compound or environmental
condition on the cells. A kinetic profile that moves from a positive peak
wavelength value to a negative peak wavelength value with increasing
concentration of the compound can indicate a negative effect of the
compound or environmental condition on the cells.

[0062] The peak wavelength values can be analyzed for kinetic profile,
wherein a kinetic profile that moves from a negative or neutral peak
wavelength value to a neutral or positive peak wavelength value over the
time course of the assay can indicate a positive or neutral effect of the
compound or environmental condition on the cells. A kinetic profile that
moves from a negative or neutral peak wavelength value to a positive or
neutral peak wavelength value with increasing concentration of the
compound can indicate a positive of neutral effect of the compound or
environmental condition on the cells.

[0064] The compound can be, e.g., a drug, a calcium channel blocker, a
β-adrenoreceptor agonist, an α-adrenoreceptor agonist, any
test reagent, a polypeptide, a polynucleotide, a modifier of a hERG
channel, or a toxin.

Methods for Reducing Risk of Drug Toxicity

[0065] One embodiment of the invention provides a method for reducing the
risk of drug toxicity in a subject, such as a human or mammalian subject.
One or more cells differentiated from an induced pluripotent stem cell
line generated from the subject can be contacted with a dose of a
pharmacological agent. The contacted one or more cells are assayed for
toxicity. The cells are applied to a colorimetric resonant reflectance
biosensor (or other biosensor) surface. The cells are contacted with the
pharmacological agent and periodic peak wavelength values are detected
during a time course of the assay. The peak wavelength values are
analyzed for frequency, amplitude, or kinetic profile or a combination
thereof over the time course of the assay. A negative change in
frequency, amplitude, or kinetic profile after the pharmacological agent
is contacted with the cells can indicate that the pharmacological agent
has a negative toxicity effect on the cells. The pharmacological agent is
prescribed or administered to the subject only if the pharmacological
agent does not have a negative toxicity effect on the contacted cells.

[0066] Another embodiment of the invention provides a method for reducing
the risk of drug toxicity in a subject, such as a human or mammalian
subject. The method comprises contacting one or more cell populations
differentiated from an induced pluripotent stem cell line generated from
the subject with two or more dose concentrations of a pharmacological
agent and assaying the contacted one or more cell populations for
toxicity. The assaying comprises applying the one or more cell
populations to a colorimetric resonant reflectance biosensor surface and
contacting the one or more cell populations with two of more
concentrations the pharmacological agent. One or more peak wavelength
values are detected for each concentration of the pharmacological agent.
The peak wavelength values are analyzed for frequency, amplitude, or
kinetic profile or a combination thereof for each concentration of the
pharmacological agent. A negative change in frequency, amplitude, or
kinetic profile after the pharmacological agent is contacted with the
cells can indicate that the pharmacological agent concentration has a
negative toxicity effect on the cells. The pharmacological agent is
prescribed or administered to the subject only if the pharmacological
agent concentration does not have a negative toxicity effect in the
contacted cells.

Methods for Screening a Compound for Neutralizing Activity

[0067] One embodiment of the invention provides methods for screening a
compound for neutralizing activity on a known toxin or negative
environmental condition (i.e., any environment condition that weakens or
kills the cells or has a negative impact on cell growth or cell
multiplication). The method comprises applying cells, cell aggregates, or
tissue to a colorimetric resonant reflectance biosensor (or other
biosensor) surface and contacting the cells, cell aggregates, or tissue
with the known toxin and a compound. Periodic or continuous peak
wavelength values are detected during a time course of the assay. The
peak wavelength values are analyzed for frequency, amplitude, or kinetic
profile or a combination thereof over the time course of the assay. A
positive change in frequency, amplitude, or kinetic profile after the
compound is contacted with the cells, cell aggregates, or tissue can
indicate that the compound has a neutralizing effect on the toxin.

[0068] Another embodiment of the invention provides methods of screening a
compound for neutralizing activity on a known toxin. The method comprises
applying one or more cells, cell aggregates, or tissue populations to a
colorimetric resonant reflectance biosensor (or other biosensor) surface
and contacting the one or more cells, cell aggregates, or tissue
populations with the known toxin and a compound at two or more compound
concentrations. Periodic or continuous peak wavelength values are
detected during a time course of the assay for each compound
concentration. Peak wavelength values are analyzed for frequency,
amplitude, or kinetic profile or a combination thereof for each compound
concentration over the time course of the assay. A positive change in
frequency, amplitude, or kinetic profile after the compound is contacted
with the cells, cell aggregates, or tissue can indicate that the compound
has a neutralizing effect on the toxin. The contacting step can further
include contacting the cells, cell aggregates, or tissue with at least
one second compound (e.g., 1, 2, 3, 4, 5, 10, 20, 30, 40, 50 or more) or
at least one second environmental condition (e.g., 1, 2, 3, 4, 5, 10, 20,
30, 40, 50 or more) in the presence of the first compound or
environmental condition.

Methods of Screening Compounds for Signature Kinetic Profiles

[0069] An embodiment of the invention provides a method of screening a
test toxin or compound for a signature kinetic profile to determine a
class or subclass of the toxin or compound. Cells, cell aggregates, or
tissue are applied to a colorimetric resonant reflectance biosensor (or
other biosensor) surface and the cells, cell aggregates, or tissue are
contacted with the test toxin or compound. Periodic or continuous peak
wavelength values are determined during a time course of the assay. The
peak wavelength values are analyzed for frequency, amplitude, or kinetic
profile or a combination thereof over the time course of the assay to
generate a signature kinetic profile of the effects of the test toxin or
test compound on the cells, cell aggregates, or tissue. The signature
kinetic profile of the test toxin or compound is compared to signature
kinetic profiles of known toxins or compounds, wherein the test toxin or
compound is placed into a class or subclass of toxins or compounds having
a similar signature kinetic profile as the test toxin or test compound.

[0070] Signature kinetic profiles are obtained by determining kinetic
profiles for two or more (e.g., 2, 3, 4, 5, 10, 15, 20, or more) classes
or subclasses of toxins (e.g., DNA damaging agents, topoisomerase
inhibitors, DNA gyrase inhibitors, RNA inhibitors, ion channel
inhibitors, etc.) or compounds. Where the two or more kinetic profiles
for toxins or compounds in the same class or subclass are similar (see,
e.g., FIG. 7), then the combination of kinetic profile is a signature
kinetic profile.

Methods of Screening for Effects on Sinus Rhythm of Cardiomyocytes

[0071] The invention provides methods for determining the effect of a test
compound or environmental condition on the sinus rhythm of
cardiomyocytes. The method compromises applying any type of
cardiomyocytes to a colorimetric resonant reflectance biosensor surface,
a dielectric film stack biosensor surface, or a grating-based waveguide
biosensor surface. The cardiomyocytes are contacted with the compound or
environmental condition and periodic or continuous peak wavelength values
or effective refractive index values are detected during a time course of
the assay. The peak wavelength values or effective refractive index
values are analyzed for sinus rhythm over the time course of the assay. A
change in the sinus rhythm after the compound or environmental condition
is contacted with the cardiomyocytes indicates that the compound or
environmental condition has an effect on the sinus rhythm of the
cardiomyocytes.

[0072] Very specific effects of compounds, combination of compounds, or
environmental conditions on sinus rhythm waves, segments and intervals
can be determined. For example, the prolongation (or shortening) of the
QT interval can be determined using methods of the invention. The heart
rate corrected QT interval, QTc, can also be determined using Bazett's
formula. Changes in length (longer or shorter) of the PR interval, PR
segment, ST segment can be determined. Additionally, widening of the QRS
complex, P wave, Q wave, R wave, S wave or T wave; abnormal deflections
of the QRS complex, P wave, Q wave, R wave, S wave or T wave; duration of
the QRS complex, P wave, Q wave, R wave, S wave or T wave; amplitude of
the QRS complex, P wave, Q wave, R wave, S wave or T wave; and morphology
of the QRS complex, P wave, Q wave, R wave, S wave or T wave (e.g., a
notch in the T wave) can be detected by the methods of the invention.

[0073] All patents, patent applications, and other scientific or technical
writings referred to anywhere herein are incorporated by reference in
their entirety. The invention illustratively described herein suitably
can be practiced in the absence of any element or elements, limitation or
limitations that are not specifically disclosed herein. Thus, for
example, in each instance herein any of the terms "comprising",
"consisting essentially of", and "consisting of" may be replaced with
either of the other two terms, while retaining their ordinary meanings.
The terms and expressions which have been employed are used as terms of
description and not of limitation, and there is no intention that in the
use of such terms and expressions of excluding any equivalents of the
features shown and described or portions thereof, but it is recognized
that various modifications are possible within the scope of the invention
claimed. Thus, it should be understood that although the present
invention has been specifically disclosed by embodiments, optional
features, modification and variation of the concepts herein disclosed may
be resorted to by those skilled in the art, and that such modifications
and variations are considered to be within the scope of this invention as
defined by the description and the appended claims.

[0074] In addition, where features or aspects of the invention are
described in terms of Markush groups or other grouping of alternatives,
those skilled in the art will recognize that the invention is also
thereby described in terms of any individual member or subgroup of
members of the Markush group or other group.

EXAMPLES

Example 1

Signature Kinetic Profiles

[0075] Vero cells were plated in complete media a 25,000 cells/well of a
colorimetric resonant reflectance biosensor microtiter plate. The cells
were exposed to one of two toxins at several different concentrations for
16 hours. The toxic effect of the toxins was evident at between 1.5 and
2.0 hours after toxin addition. The IC50 for Toxin X was 37 ng/ml.
The IC50 for Toxin Y was 0.187 ng/ml. The kinetic profile of Toxin X
and Toxin Y is shown in FIG. 1. There is a concentration dependant
negative shift in PWV as the toxins kill the cells.

[0076] In another experiment, Vero cells were again plated into wells of a
colorimetric resonant reflectance biosensor microtiter plate. Two toxins
were added to the cells, Toxin X or Toxin Y. Compound 4 or Compound 1
were also added to the wells. Compound 4 blocks Toxin X, but does not
block Toxin Y. Compound I blocks both Toxin X and Toxin Y. In wells where
Compound 4 was added Toxin X was blocked. FIG. 2A shows the blocking of
cell death by Toxin X over time (lighter lines). Cell death caused by
Toxin Y was not blocked as demonstrated by a shift to negative PWVs. See
FIG. 2A, darker lines. Where Compound 1 was added to the cells, cell
death by the toxins was blocked by Compound 1 and a neutralization of
negative PWVs is seen. See FIG. 2B. Therefore, assays of the invention
can be used to screen for compounds that neutralize toxins.

[0077] FIG. 3A-B demonstrates an experiment where increasing
concentrations of toxin were mixed with neutralizing doses of an
antidote. CHO cells were plated at 25,000 cells/well on a CA2 384-well
BIND® biosensor plate in complete media for 3-4 hours. A toxin or a
toxin:antidote mixture was then added to the wells. Cells were monitored
using a BIND® biosensor plate reader for 15-16 hours at room
temperature. The results are shown in FIG. 3A-B. FIG. 3A shows the
temporal response profile and FIG. 3B shows the results at an 11 hour
time point. The antidote protects cells from cell death as indicated by
neutralization of the dose-dependent, negative PWV shift elicited by
toxin.

[0078] In another experiment HeLa cells were treated with 100 uM of
tamoxifen (a calcium influx stimulator), doxorubicin (a DNA damaging
agent), cycloheximide (a protein synthesis inhibitor), digitonin (a mild
detergent), and a buffer, and monitored every 15 minutes on a BIND®
Reader for 40 hours in a 37° C. incubator. The cytotoxic compounds
each have distinct mechanisms of action that have distinct kinetic
profiles in the assays of the invention. See FIG. 4. Toxins can be tested
for their effect on differing types of cells and can be placed into a
class or sub-class of toxins (e.g. calcium influx stimulator) based on
their kinetic profiles. These assays have a higher throughput than
electrical impedance testing for screening and profiling of toxic
compounds. FIG. 5 shows the PWV shift for each of the cytotoxic agents in
relation to the concentration of the cytotoxic agent (FIG. 5A:
cycloheximide; FIG. 5B: digitonin; FIG. 5C: doxorubicin; FIG. 5D:
tamoxifen).

[0079] The cytotoxic activity of tamoxifen is thought to occur by inducing
calcium mobilization. The kinetic profile of tamoxifen includes a sharp
downward shift in PWV at early time points. See FIG. 6.
4-hydroxy-tamoxifen is a metabolite of tamoxifen, with higher affinity
for estrogen receptor and greater toxicity. 4-hydroxy-tamoxifen has a
kinetic profile that has a faster onset of toxicity than tamoxifen. See
FIG. 6. Raloxifene (unlabeled line) is from the same class (SERM, or
estrogen receptor modulators) as tamoxifen, but has dramatically reduced
side effects and reduced cytotoxicity. The kinetic profile of raloxifene
demonstrates lack of cytotoxic response. See FIG. 6.

[0080] FIG. 7 shows the PWVs for several known DNA damaging agents over
time. All have same basic profile of a sudden onset, steep decline,
followed by a negative PWV plateau. Cisplatin (intercalating agent),
potassium dichromate (intercalating agent), doxorubicin (crosslinking
agent), and mitomycin (crosslinking agent) all have similar profiles and
all are direct DNA damaging agents. Camptothecin is a topoisomerase
inhibitor and has somewhat different kinetic profile. FIG. 8A-B shows the
PWVs for differing concentrations of potassium dichromate (FIG. 8A) and
cisplatin (FIG. 8B).

[0081] Many compounds have undesired effects on microtubules. Vinblastine
binds to tubulin and disrupts microtubule formation. The kinetic profile
of vinblastine is distinct from other toxins. See FIG. 9A-B. The earlier
time points (FIG. 9B) show a rapid acute response and then a gradual
negative PWV shift over the long term. The early response is likely a
result of acute morphological effects elicited by microtubule disruption.
The longer-term negative PWV response is likely the result of cell death
(FIG. 9A). This assay highlights the potential of multiple readouts (on
target+toxicity) in one assay.

Example 2

Frequency and Rate Determinations

[0082] mES-derived cardiomyocytes (Cor.At) were obtained from
Axiogenesis/Lonza. 5,000 cells per well were plated on fibronectin-coated
384 well biosensor plates for 24 h before experiment. These cells beat in
culture. Cells were treated with doxorubicin for 17 h at 37° C.
with constant monitoring using the BIND® Reader. In FIG. 10A the
buffer reading demonstrates the beating of the cells in culture. There is
an oscillation of PWVs that indicates the beating of the cells. The
amplitude of the beating becomes stronger over time. That is, there is Y
axis spread of the PWV reading grows larger over time as the cells beat
stronger in culture. Where doxorubicin is added to the cells, the PWV
readings become negative. Additionally, the amplitude of the cells
becomes weaker over time. The effect of doxorubicin is dose dependant.
See FIG. 10B.

[0083] Murine embryonic stem cell-derived cardiomyocytes were added to
wells of a biosensor and a BIND® Reader recorded PWVs at the rate of 4
reads per second. One well was treated with KCl and another well was not
treated. The results are shown in FIG. 11. The cells beat synchronously
when cultured on BIND® optical biosensors and the frequency of the
beating can be detected. See FIG. 12. KCl treatment leads to a loss of
amplitude of the beating and decrease in the frequency of the beating.
The BIND® Reader detects beating frequency and rate as oscillations of
positive-negative PWV shifts. Kinetic PWV profiles can be used to
accurately measure beating rate and frequency. Therefore, the assay
provides an ultra high-throughput assay to measure off-target drug
effects on cardiotoxicity and contractility.

[0084] In another experiment the cardiomyocytes were plated at 20,000
cells per well on fibronectin coated biosensor plates. The cells were
incubated for 72 h at 37° C. before the experiment. The cells were
monitored for 3 minutes before KCl was added to 50 mM final
concentration. The results are shown in FIG. 13. The rate and frequency
of the beating of the cells were drastically reduced upon addition of the
KCl to wells. This demonstrates that the monitoring of cells before
compound addition and after compound addition can be done in a single
well.

[0085] In another experiment cardiomyocytes were treated for 17 hours with
several different concentrations of doxorubicin (10 μM, 1 μM, 0.1
μM, 0.01 μM, and 0 μM). The beating amplitude and frequency was
measured at 4 reads/second prior to doxorubicin treatment, then again
after the 17 hour doxorubicin incubation. The results are shown in FIG.
14. The amplitude and frequency of beating is disrupted by doxorubicin in
a concentration-dependent manner.

Example 3

[0086] An Ocean Optics HT2000+ spectrometer in a BIND® Cartridge Reader
was altered so that the slit that allows light into the spectrometer was
widened, allowing more light to enter the enter the spectrometer.
Therefore, recordings of up to 1000 Hz, i.e. up to 1000 readings/second
can be obtained when the BIND® Cartridge Reader is "parked" (i.e., the
BIND® Cartridge Reader remains in one position over, e.g., a well
holding cells). The recordings are made continually in real-time.
Recordings can be taken at about 2, 4, 10, 50, 80, 100, 250, 280, 300,
400, 500, 600, 700, 800, 900, 1,000 or more Hz (or any range between
about 2 and about 1,000 Hz).

[0087] The refined BIND® Cartridge Reader was used to take measurements
of cardiomyocyte beating. FIG. 15 shows readings taken at 80 Hz. By
comparison, the data in Examples 1 and 2 was measured at 4 Hz. The
increased sampling rate allows a much more refined look at the shape of
each individual beat. Different beating "phenotypes" can be determined
from well-to-well, in addition, the difference between synchronous
beating across the well and asynchronous beating can be determined. FIG.
15A shows high frequency beating, FIG. 15B shows moderate frequency
beating, FIG. 15C shows slow frequency beating, FIG. 15D shows irregular
frequency beating. FIG. 16A-16B shows dense synchronous beating. FIG.
16C-D shows sparse asynchronous beating. The effects of compounds that
have cardiotoxic properties, such as blocking hERG channels and/or affect
QT prolongation can be examined. The QT interval is a measure of the time
between the start of the Q wave and the end of the T wave in the heart's
electrical cycle. A prolonged QT interval is a risk factor for
ventricular tachyarrhythmias and sudden death. Methods of the invention
can be used to screen for compounds that result in QT prolongation in
moderate/high throughput screens. The methods of the invention can
measure subtle-to-significant effects on the beat of cardiomyocytes in
culture, which are predictive of effects of beating hearts in vivo.

[0088] Amitriptyline is a tricyclic antidepressant sold under the trade
name Elavil®. Amitriptyline functions primarily as a
serotonin-norepinephrine reuptake inhibitor by modulating transporters
for both transmitters. It is associated with heart arrhythmias due to
hERG channel modulation and QT prolongation. Amitriptyline was added to
cardiomyocytes that were beating in culture and PWVs were constantly
monitored. FIG. 17A shows the PWVs of the cells over time prior to the
addition of the amitriptyline. FIGS. 17B, 17C, and 17D show the PWVs of
the cardiomyocytes over time at 6 minutes, 10 minutes, and 15 minutes,
respectively, after the addition of amitriptyline. The change from
regular, synchronous beating to irregular, non-synchronous beating can
clearly be seen after the addition of the amitriptyline.

[0089] Therefore, methods of the invention can be used to screen the
effect of compounds, a combination of compounds, or environmental
condition on the kinetic profile, beating frequency and beating rate of
cardiomyocytes and other cells.

Patent applications by Alexander Yuzhakov, West Roxbury, MA US

Patent applications by Eric Sandberg, Dupont, WA US

Patent applications by Rick Wagner, Cambridge, MA US

Patent applications by Steven Shamah, Acton, MA US

Patent applications in class Oxygen of the saccharide radical bonded directly to a polycyclo ring system of four carbocyclic rings (e.g., daunomycin, etc.)

Patent applications in all subclasses Oxygen of the saccharide radical bonded directly to a polycyclo ring system of four carbocyclic rings (e.g., daunomycin, etc.)